The long-term objective of this project is to understand the mechanism by which phosphorylation of alpha-synuclein (1-syn) enhances the toxicity of this neuroprotein. Employing a yeast system, we seek to extend our findings that a highly conserved phosphatase and two highly conserved signaling proteins prevent 1-syn-induced ROS and cell death. The specific aims are to: 1) Determine whether ROS originate from mitochondria or peroxisomes by expressing 1-syn in respiratory-deficient cells or peroxisome-deficient cells and staining with an ROS-sensitive dye. 2) Determine the mechanism by which phosphorylation of 1-syn at S129 enhances 1-syn's ability to cause cell death. A two-hybrid screen will be employed to find proteins that interact with the toxic phosphorylated form of WT 1-syn. 3) Characterize how cells die when the kinase/phosphatase balance shifts in favor of kinases. Although the essential phosphatase that we discovered that protects cells from 1-syn-induced ROS cannot be deleted, its non-essential regulatory subunits can be. Experiments will use GFP-tagged 1-syns to determine whether 1-syn inclusion formation, proteasome dysfunction, or mitochondrial dysfunction causes cell death when the kinase/phosphatase balance is tipped in favor of the kinases by knocking out phosphatase regulatory genes. 4) We discovered two yeast signaling genes that exhibit synthetic lethal interactions with WT 1-syn and A30P but not A53T. These signaling genes are also present in human neurons where they regulate the cell cycle, intracellular signaling, differentiation, ion-channels, vesicle trafficking, and apoptosis. We hypothesize that these two signaling proteins bind to toxic phosphorylated forms of 1- syn (WT or A30P), and this protects cells from the build up of 1-syn. This hypothesis will be tested by monitoring for direct binding between the various 1-syns and the signaling protein and by monitoring how cells die when the signaling genes are deleted. Understanding how kinases/phosphatases and signaling molecules regulate 1-syn phosphorylation state and hence its toxicity could lead to novel neuroprotective therapeutics that could delay or even prevent the onset of PD. Such therapeutics would modulate the kinase-phosphatase equilibrium in such a way as to drive 1-syn into a dephosphorylated state.7. Project Narrative A simple chemical modification (phosphorylation) of the Parkinson's disease-related protein alpha- synuclein dramatically increases the toxicity of alpha-synuclein. Our goal is to determine the enzymes and proteins that regulate the chemical modification of alpha-synuclein. This information could lead to novel drugs that function to inhibit the chemical modification of alpha-synuclein.